Mitochondrial division inhibitor (Mdivi-1) and CaMKII inhibitor (KN-93) attenuate Ipratropium Bromide mediated myocardial injury in in vitro models of myocardial Ischaemia/Reperfusion

  • Fatmah Abubaker Ahmed Khalefah

    Student thesis: Doctoral ThesisDoctor of Philosophy


    Introduction: A common background links ischaemic heart disease (IHD) with chronic obstructive pulmonary disease (COPD) with regards to systemic inflammation and underlying morbidity and mortality due to myocardial infarction (MI). The symptoms and clinical manifestations of both COPD and IHD are similar, however, compounds that are designed to target both diseases are yet to be fully evaluated. For patients with COPD and IHD who are treated, there is much evidence that muscarinic antagonists, such as ipratropium, whilst acting as effective bronchodilators also pose the risk of adverse cardiovascular events, such as myocardial infarction and stroke, due to underlying cardiotoxic effects of these bronchodilatory drugs. According to the World Health Organization (WHO) 2019 statistics, COPD is considered to be a disease which highly contributes to mortality as well as morbidity across a wide-range of socioeconomic economies with a considerable increase in the burden and impact on global health. Previous work in our laboratory has shown that Ipratropium Bromide (Ip), a non-selective muscarinic receptor antagonist prescribed for the treatment of COPD, exacerbates myocardial injury. After administration during reperfusion in clinically relevant in vitro models of ischaemia/reperfusion (I/R) for the heart muscular (left ventricular) and hypoxia/reoxygenation (H/R) for isolated cardiac myocytes. The injury was via triggering both apoptosis, necrosis and increase the infarction of the heart tissue significantly, in a dose dependent mechanism. Ip’s mechanism of action in I/R and H/R was measured by determining the effect of the drug treatment during these conditions and to further gain an insight of its action. Despite evidence to suggest that the cytotoxic effect of administrating Ip during reperfusion might involve mitochondrial fission and the opening of mitochondrial permeability transition pore (mPTP) as well as altered calcium signalling, the mechanism of this drug-induced toxicity is yet to be fully elucidated.

    Aims: The aim of this work was to try to further elucidate the mechanisms which lead to Ip induced myocardial injury, with a specific focus on the role of Ca2+ signalling and mitochondrial involvement. By investigating the cytotoxic effects of Ip in in vitro models, such as Langendorff perfused hearts as well as the cytotoxic effects in adult cardiac myocytes in stressed conditions which were clinically relevant to myocardial I/R.

    Methods: Multiple methods were employed in order to identify of the mechanisms of toxicity for Ip and associated signalling pathways. Langendorff, Western Blotting, flow cytometry and PCR were all carried out under simulated conditions of I/R and H/R for the isolated rat hearts and cardiac myocytes in order to identify if there was a mitochondrial role in Ip induced cardiotoxicity. This was primarily by using mitochondrial division inhibitor (Mdivi-1), which has inhibition properties for cell death as well as providing protection against heart failure progression, the protective effects of mdivi-1 is thought to be by preventing mitochondrial fragmentation and blocking pro-apoptotic BAX-dependent cytochrome c release. And using KN-93 which can be defined as an inhibitor with a selectivity and a high potency against CaM kinase II, KN-93 works in the cardiomyocytes via maintaining the homeostasis of calcium, to confirm the involvement of calcium overload as a calcium signalling indicator. As well as trying to identify whether Ip increases the expression of Dnm1l, which is Dynamin-1-like protein the gene encoded GTPase for Drp1. Drp1 is a member of the conserved dynamin large GTPase superfamily which regulates mitochondrial fission functions. As Ip significantly increases infarction, apoptosis, caspase-3 activity, the expression of Akt these have been preciously shown to be correlated with a reduction in the cell viability following I/R injury.

    Results: One of the main findings in this study is that the administration of Mdivi-1 provides cardio-protection when co-administered with Ip at both 15 and 30 minutes after reperfusion by the reduction in Dynamin related protein 1 (Drp-1), Akt and Erk1/2 when compared with the untreated control. Moreover, this study also showed it can reduce myocardial injury exacerbated by Ip via reduction in the infarct size significantly by (82.7 ± 2.9% vs. 158.5 ± 6.2%) (p <0.001) and total apoptosis as shown through the flow cytometry caspase-3 activity and it also significantly increased cardiac myocyte viability. The effect that occurred on Akt and Erk1/2 might be a result due to the action of Mdivi-1 on the mitochondria, indicating protection, although by a mechanism which has not previously been described and remains to be fully characterised.

    Similar results were found in the KN-93 studies with protective properties to cardiac myocytes in all cellular models used. KN-93 mainly reduced the infarction size by (74 ± 4.4% vs. 100 ± 0) (p <0.001). as well as caspase-3 activity, significantly. This is assumed to be mainly by inhibiting CaMKII which also inhibits calcium overload. Since calcium overload resulted in the cell death as found by the study, confirming the involvement of calcium signaling and, again, indicating involvement of the mitochondria which is linked to calcium overload.

    Combining the obtained results, the injury exacerbated by Ip was attenuated by both Mdivi-1 and KN-93 when co-administered with Ip, both agents have shown significant reduction in the injury when co-administered with Ip in infarction via the measurement of AAR% using Langendorff, TTC staining technique data were normalized to I/R control. caspase-3 activity usig flow cytometry for isolated cardiac myocyrtes stimulated H/R and addition of treatment stain to measure caspase-3 activity, data were normalized to H/R control. Mdivi-1 provided a higher level of protection since it decreased apoptosis and increased cell viability which, while there was attenuation, was not seen in KN-93 results.

    In conclusion: The injury caused by Ip is potentially due to both calcium overload and phosphorylation of Drp1, although further studies are required to fully understand the mechanism of action and the full pathway. This work provides further elucidation into the mechanism by which ipratropium elicits its cardio-toxic effects and this holds promise for the development for future treatments for both COPD and IHD.

    Date of AwardApr 2022
    Original languageEnglish
    Awarding Institution
    • Coventry University
    SupervisorKate Harvey (Supervisor), Bernard Burke (Supervisor) & Afthab Hussain (Supervisor)

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